1. Field of the Invention
The present invention relates generally to the field of molecular biology, and more specifically to veterinary clinical laboratory diagnostic methods. In particular, a system, method, compositions, and diagnostic kits are provided for identification, amplification, quantitation and/or detection of nucleic acid segments derived from specific viral species, types, and/or subtypes, including those species implicated as causal or contributory agents in bovine respiratory disease complex (BRDC). In particular, methods and compositions are disclosed for identifying and differentiating between the three known subgenotypes of Bovine Viral Diarrhea Virus (BVDV).
2. Description of Related Art
“SHIPPING FEVER”
“Shipping fever” is a term given to an acute, highly-contagious, septicemic syndrome in cattle and sheep that is characterized clinically by fever, acute inflammation of the airways, nasal discharge, anorexia, depression, fibrinous pneumonia, and necrosis of the infected tissues. Most frequently encountered in feedlots following shipping, shipping fever is the major cause of death among young cattle, and is responsible for an estimated annual loss to the industry of more than half a billion dollars. In 1991 alone, shipping fever was estimated cost the U.S. cattle industry almost $624 million, due primarily to the costs of treatment, production loss, and death.
The pathogenesis of shipping fever is generally considered to involve adverse external influences predisposing the animal to an initial viral respiratory infection, which, in turn, produces conditions favorable for the proliferation of one or more secondary bacterial infections.
Bovine Respiratory Disease (BRD) Complex (BRDC)
BRDC (often commonly referred to as “shipping fever.”) is a multifactorial disease complex that frequently afflicts stocker/feeder calves in market channels and at destination pasture or feedlot. Characterized by concomitant sequential or simultaneous infection by both viral and bacterial pathogens, BRDC is a major cause of economic loss in the beef and dairy cattle industries.
The disease, which infects cattle of all ages (including nursing calves), is characterized by rapid breathing, coughing, depression, loss of appetite, ocular and nasal discharge, and elevated temperatures. In an acute outbreak, death may follow within 24 hours of onset of symptoms. Calves exhibiting depression will have drooping ears, an extended head, a bowed back and/or often isolate themselves from other cattle. As the health of the calves progressively deteriorates, they stop feeding, exhibit an increased respiratory rate, and develop a pronounced fever (typically in the 104°-108° F. range).
At least four viral pathogens have been implicated in BRDC. They include bovine herpes virus 1 (BHV-1) (the causal agent of infectious bovine rhinotracheitis [IBR]), bovine parainfluenza type 3 virus (PI3), one or more genetic variants of bovine viral diarrhea virus (BVDV), and Bovine respiratory syncytial virus (BRSV).
Following viral infection, the affected animals then develop one or more subsequent bacterial infections (e.g., Mannheimia [formerly Pasteurella] haemolytica, Pasteurella multocida, Histophilus somni (formerly Haemophilus somnus), Actinomyces pyogenes, and various Mycoplasma spp.) which often manifest in acute pneumonia.
Bovine Viral Diarrhea Virus (BVDV)
BVDV is now recognized as an important etiologic agent in BRDC. Spread through a herd in a fecal-oral manner, the virus causes acute infections (including diarrhea, fever, and hemorrhagic syndrome), and is capable of crossing the placenta of pregnant cattle, which often results in the birth of persistently infected (PI) calves that are immunotolerant to the virus and persistently viremic for the rest of their lives. PI bovids currently represent the major reservoir of the virus; these animals are highly predisposed to infection with microorganisms causing pneumonia or enteric disease, and provide the necessary viral reservoirs for outbreaks of fatal mucosal disease (MD) in cattle (see e.g., Liess et al., 1974; Barber et al., 1985; Olafson et al., 1946; Ramsey et al., 1953; Malmquist, 1968; and Ross et al., 1986, each of which is specifically incorporated herein in its entirety by express reference thereto).
Bovine Viral Diarrhea viruses are a disparate group of viruses that can be classified both phenotypically (see, e.g., Baker, 1995) (cytopathic or noncytopathic) and genotypically (see, e.g., Ridpath et al., 1994; Vilcek et al., 2001; and Flores et al., 2002). Establishing protective immunity against BVDV in livestock has been problematic for a number of reasons. As in some other virally-mediated diseases, the levels of serum antibodies against BVDV do not necessarily correlate with protection against disease. Establishing protective immunity in nursing calves presents additional obstacles, since maternal antibodies to BVDV may deplete the injected immunogen and effectively neutralize the vaccine.
A study by Fulton et al. (2006) evaluated 21,743 calves entering U.S. feedlots and determined that 88 calves were persistently infected (PI) with BVDV. Of the 88 PI calves, 77.9% were infected with BVDV-1b; only 11.6% were infected with BVDV-1a, and only 10.5% were infected with BVDV-2a.